Ectodermal organ development is initiated by inductive tissue interactions. Developing teeth, epidermis, hair, and limbs are classic examples of such inductive processes. Tooth development can be divided into the initiation, bud, cap, and bell stages. In mice, tooth development begins at E11.5 by thickening of the dental epithelium. The dental lamina undergoes further proliferation and subsequently develops into the tooth bud and germ. The tooth bud is formed by the invagination of the placode and the condensation of mesenchyme cells adjacent to the bud. At the cap stage (E14.5), dental epithelial cells differentiate into several cell types, such as the inner dental epithelium and enamel knot cells. Cell death by apoptosis within the enamel knot is critical for cusp formation in molars. At the bell stage (E17.5), the dental mesenchyme differentiates into dentin matrix-secreting odontoblasts, and then the inner dental epithelial cells differentiate into enamel matrix-secreting ameloblasts. The goal of the project is to discover novel and previously uncharacterized genes to understand how tooth and craniofacial tissues develop and to define molecular defects underlying anomalies of these tissues. [unreadable] [unreadable] We previously identified ameloblastin (Ambn) as a new member of the enamel matrix proteins. We found that Ambn was secreted by ameloblasts, localized at the apical region of the cells, and served as the adhesion molecule for ameloblasts. When odontoblasts differentiate from the dental mesenchyme, the basement membrane separating the dental mesenchyme and epithelium is replaced by predentin. However, the matrix molecule responsible for odontoblast attachment to predentin is unknown. The new dentin matrix molecule we discovered, TM14, may serve this role. We initially identified TM14 as a gene preferentially expressed in developing teeth by differential hybridization using tooth germ cDNA microchips. The TM14 protein has EGF modules, a fibulin motif, and a unique N-terminal domain and can be classified as a new member of the fibulin family: fibulin-7. TM14 mRNA was expressed in preodontoblasts and odontoblasts. TM14 bound preferentially to dental mesenchyme cells and odontoblasts. We demonstrated that the protein also interacted with heparin, fibronectin, fibulin-1, and dentin sialophosphoprotein. Immunostaining revealed that TM14 was localized at the apical pericellular regions of preodontoblasts. When the dentin matrix was fully formed, TM14 was localized in predentin and dentinal tubules. These results suggest that TM14 plays roles in odontogenesis and dentinal tubule structures. [unreadable] [unreadable] We previously identified epiprofin (Epfn)/Sp6 as a zinc-finger transcription factor that is expressed in certain developing ectodermal tissues such as teeth, hair follicles, skin, and limbs. We created Epfn knockout mice (Epfn-/-) to identify its in vivo function. Epfn-/- mice had defects in tooth and developed a thick skin, hairlessness, and digital fusions. Mutant incisors and molars erupted with excess numbers and had severe enamel defects. Mutant mice have defects in cusp and root formation and abnormal dentin structure. In Epfn-/- mice, a single dental placode and bud formed at the initial stage. However, later, multiple branches of the dental epithelium had invaginated into the dental mesenchyme. In Epfn-/- teeth, rapid proliferation and differentiation of the inner dental epithelium were inhibited, and the dental epithelium retained the progenitor phenotype. Multiple premature non-proliferating enamel knot-like structures were formed ectopically. We found that transfection of an Epfn vector promoted dental epithelial cell differentiation and activated promoter activity of the ameloblastin gene, suggesting that Epfn promotes ameloblast differentiation. Our results suggest that in Epfn-deficient teeth, ectopic, non-proliferating regions likely bud off from the self-renewable dental epithelium, form multiple branches, and eventually develop into supernumerary teeth. Thus, Epfn has multiple functions for cell fate determination of the dental epithelium by regulating both proliferation and differentiation, preventing continuous tooth budding and generation.